SIMPLE ELECTROCOMPETENT CELLS -FROZEN
YETP: 0.75% Bacto yeast extract, 1.00% Bacto tryptone
10%
Glycerol (v/v) filter sterilized
sterile water
Day 0 or 1
Streak out ccdB survival cells ( should work if you
start in the AM early )
Pick colony and grow overnight, in LB.
Pull O.N. out in morning and let sit on bench,
Place 2 500 ml cultures in 20¡C shaker at 250 rpm to
cool to 20¡C .
Innoculate with 0.5 ml overnight at noon. Note that the dilution required to get
mid log cells the next morning may differ if not using ccdB survival cells.
Next morning collect cells at 9 am should be ~ OD
600=0.6 to 0.8
Chill cells on ice for 15 minutes
Spin down cells 10
minutes @ 5500 rpm in SLA1500 at 4¡C.
Resuspend cells in
20 ml, dilute to 200 ml cold water
Spin, 10 min at
5,000 rpm in SLA1500
Resuspend cells in
20 ml, dilute to 200 ml cold water
Spin, 10 min at
5,000 rpm in SLA1500
Resuspend washed
cells in total of 50 ml of cold 10% glycerol
Combine cells into
single centrifuge tube
Spin down cells
and resuspend in 8ml 10% glycerol
Dispense
cells into 100 µl aliqouts and freeze in liquid nitrogen. Store at Ð80¡C.
Day 4
titer cells using 0.1 ng of a standard plasmid.
Electroporation Protocol
1.
Place
cuvettes(s) and ependorf tubes on ice.
2.
Thaw electrocompetent cells on ice. Dive 100 µl aliquot into two tubes if
needed.
3.
Add
ligation, gateway reaction, or plasmid DNA to cells ( 1-3 µl) and gently mix by
pipetting. Note that salt will be
a major problem. Only use a few µl
of ligation mix. Incubate on ice for a few minutes and transfer cells to the
cuvette.
4.
Zap
cells in the Salkoff lab Biorad Electroporator. Set apparatus to 2.5 kV, 25 µF, 200 ohms. Cuvette holder is in the Ð20¡C freezer.
5.
Immediately
add 1 ml LB to cuvette.
6.
Transfer
cells to an ependorf tube and incubate at 37¡C for 30 to 60 minutes.
7.
Plate
100 ul (and/or rest after spinning down cells.
NOTES:
Good
high efficiency cells depends on keeping everything cold. I just do all the work in the cold room
so I know nothing will warm up.